Recurrent infection and deterioration of lung function are the major causes of morbidity and mortality in cystic fibrosis (CF). Although various mechanisms relating defective CFTR function to CF lung disease have been proposed, the two principal hypotheses that have emerged are: Na+ hyperabsorption/airway surface liquid (ASL) dehydration and defective airway submucosal gland function. We will use novel measurement methods and model systems to define the role of submucosal glands and airway surface epithelium in CF lung disease, and their correction by small-molecule activators of AF508-CFTR. In Aim 1, the mechanisms of defective airway submucosal gland function in CF lung disease will be defined. We propose that impaired submucosal gland fluid and HCO3- secretion in CF airways results in a hyperviscous and acidic ASL, which promotes bacterial colonization and CF lung disease. Ion (Na+, CI-, HCO3-) and water transport will be measured in glands in intact, non-CF and CF human airways, partially microdissected/exposed glands, and primary cultures of gland serous and mucous cells. The second part of Aim 1 will link defective gland function to CF lung disease, using collected non-CF vs. CF gland fluid. In Aim 2, we will determine the contributions of defective gland vs. airway surface function to ASL abnormalities in CF. We will critically examine the 'ASL dehydration'hypothesis using human airways, testing whether ASL volume is reduced in CF, how ASL volume is regulated in intact airways, and the role of submucosal glands vs. surface epithelium in regulating ASL volume. In Aim 3, small-molecule correctors of AF508-CFTR will be used to test whether restoration of CI- transport in CF epithelia corrects gland/ASL defects in CF. It has been assumed that replacing (by gene therapy) or restoring (by small-molecules) CFTR CI- transport in CF will be beneficial in preventing lung disease. This aim follows from our discovery of small-molecule correctors of defective gating and cellular processing of AF508-CFTR. Novel, fluorescence-based assays in airway spheroids and primary cell cultures will be used to evaluate small-molecule efficacy in restoring epithelial cell CI- conductance. Gland function and ASL properties will be measured in small-molecule-treated cell cultures and ex vivo tissues. The data will provide a rigorous scientific rationale for use of small-molecules in CF therapy, and establish widely useful assays to prioritize small molecules for clinical development.